In humans, mutations in dehydrogenase E1 and transketolase domain containing 1 (DHTKD1) are associated with neurological abnormalities and accumulation of 2-oxoadipate, 2-aminoadipate, and reactive oxygen species. The protein encoded by DHTKD1 has sequence and structural similarities to 2-oxoglutarate dehydrogenase, and the 2-oxoglutarate dehydrogenase complex can produce superoxide/H₂O₂ at high rates. The DHTKD1 enzyme is hypothesized to catalyze the oxidative decarboxylation of 2-oxoadipate, a shared intermediate of the degradative pathways for tryptophan, lysine and hydroxylysine. Here, we show that rat skeletal muscle mitochondria can produce superoxide/H₂O₂ at high rates when given 2-oxoadipate. We identify the putative mitochondrial 2-oxoadipate dehydrogenase complex as one of the sources and characterize the conditions that favor its superoxide/H₂O₂ production. Rates increased at higher NAD(P)H/NAD(P)⁺ ratios and were higher at each NAD(P)H/NAD(P)⁺ ratio when 2-oxoadipate was present, showing that superoxide/H₂O₂ was produced during the forward reaction from 2-oxoadipate, but not in the reverse reaction from NADH in the absence of 2-oxoadipate. The maximum capacity of the 2-oxoadipate dehydrogenase complex for production of superoxide/H₂O₂ is comparable to that of site I_F of complex I, and seven, four and almost two-fold lower than the capacities of the 2-oxoglutarate, pyruvate and branched-chain 2-oxoacid dehydrogenase complexes, respectively. Regulation by ADP and ATP of H₂O₂ production driven by 2-oxoadipate was very different from that driven by 2-oxoglutarate, suggesting that site A_F of the 2-oxoadipate dehydrogenase complex is a new source of superoxide/H₂O₂ associated with the NADH isopotential pool in mitochondria.